The following relates to the imaging arts. It is described with particular reference to example embodiments that relate to imaging of rare cells, such as epithelial cells, in the buffy coat of a centrifuged blood sample. However, the following relates more generally to illumination systems for generating a substantially uniform static illumination across a large field of view and to microscopes employing same.
In the technique of quantitative buffy coat analysis, a whole blood sample is drawn and processed using anti-coagulant additives, centrifuging, and so forth to separate the blood into components including a buffy coat component comprised principally of white blood cells. Rare cells of interest which are present in the buffy coat, such as certain epithelial cells associated with certain cancers, are tagged using a suitable fluorescent dye, and fluorescence microscopic imaging is then used to count the fluorescent dye-tagged cells of interest. Quantitative buffy coat analysis is a promising non-invasive technique for screening for certain cancers, for monitoring cancer treatment, and so forth.
The concentration of fluorescent dye-tagged rare cells in the buffy coat is low. Optical scanning fluorescence microscopy enables assessment of a large area of buffy coat sample by scanning a field of view of a microscope relative to the buffy coat sample. Scanning can be achieved by moving the microscope relative to the buffy coat sample, by moving the buffy coat sample relative to the microscope, or by some combination thereof. A large field of view illuminated with high intensity uniform light is advantageous for rapidly and accurately assaying the fluorescent dye-tagged rare cells in the buffy coat sample. The illumination may also advantageously employ monochromatic or narrow-bandwidth light so as to facilitate spectral differentiation between rare cell fluorescence and scattered illumination.
However, providing illumination at high intensity that is uniform over a large field of view is difficult.
In the case of white light sources, filtering is typically required to provide monochromatic or at least spectrally restricted illumination. Spectral filtering blocks a large portion of the optical output that lies outside the selected spectral range. Thus, illuminating with a white light source is optically inefficient. High intensity incandescent white light sources such as Xenon lamps also produce substantial heat, which can adversely affect the quantitative buffy coat analysis.
A laser light source is more optically efficient at producing spectrally narrow light. For example, an argon laser outputs high intensity narrow spectral lines at 488 nm and 514 nm, and weaker lines at other wavelengths. These wavelengths are suitable for exciting luminescence in certain tagging dyes that luminesce at about 550 nm.
However, lasers typically output a tightly collimated beam having a highly non-uniform Gaussian intensity profile or distribution across a narrow beam cross-sectional area. Moreover, the laser beam is coherent and typically exhibits a speckle pattern due to interference amongst the wave fronts. The speckle pattern can have spatial frequencies that overlap the typical size of rare cells. The speckle pattern can also shift or change as the field of view is scanned. These aspects of laser light substantially complicate determination of whether a detected luminous feature is a fluorescent dye-tagged rare cell, or an illumination artifact.
Spatial uniformity can be improved using a beam homogenizer. One type of beam homogenizer operates by providing an inverse Gaussian absorption profile that substantially cancels the Gaussian beam distribution. Another type of beam homogenizer employs two or more lenses (or a compound lens) to refract the Gaussian beam in a way that redistributes the light into a flattened spatial profile. Using a beam homogenizer in microscopic fluorescence imaging is problematic, however, because focusing of the homogenized beam by the microscope objective can introduce additional beam non-uniformities. Moreover, beam homogenizers generally do not substantially reduce speckle non-uniformities.
In another approach, known as confocal microscopy, the laser beam is rapidly rastered or scanned across the field of view. The field of view is sampled rather than imaged as a whole. In this dynamic approach, the portion of the sample illuminated at any given instant in time is much smaller than the field of view. By rapidly rastering the focused laser beam over the field of view, an image can be constructed from the acquired sample points. A uniform illumination is, in effect, dynamically simulated through rapid sampling of the field of view.
Confocal microscopy is an established technique. However, the beam rastering adds substantial complexity and cost to the microscope system. Confocal microscopy can also be highly sensitive to small defects in a lenses or other optical component. Thus, very high quality optics should be employed, which again increases system cost.